Methods for staining cells for identification and sorting

ABSTRACT

The present invention provides novel methods of cell staining, such as bovine sperm, using electroporation or osmolality treatments at viability-enhancing temperatures. Furthermore, methods of highly efficient cell sorting that are especially suitable in sorting bovine sperm using novel cell staining procedures are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application incorporates by reference the related application,METHOD AND APPARATUS FOR SORTING CELLS, Ser. No. ______, filed ______,and Representative Docket Number 089000-0138.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

FIELD OF THE INVENTION

The invention relates to techniques and systems for visualizing livecells using novel staining procedures.

BACKGROUND OF THE INVENTION

The in vivo identification of a target cell population is required, andrequired quickly, in many industries. Such applications include thosewhere the selected cells are destined for other applications thatrequire the cells to be living after identification. For example, cellsare processed using fluorescence-activated cell sorting, where cells arecultured and expanded in vitro after sorting, or in sperm sorting bygender in animal husbandry applications.

Being able to pre-select animal offspring gender would allow moreefficient operations of livestock producers. Dairy farmers have littleuse for most bull calves. For example, males are preferred in beefcattle and sheep because males grow faster, producing more meat morequickly.

The male reproductive cells, the sperm, determine the gender of theoffspring. Most males carry an X and a Y sex chromosome, whereas femalescarry two X chromosomes. A sperm or an egg contains one half of thatparent's genetic information; however, the egg only carries an Xchromosome one of each pair of autosomes. In mammals, the egg alwayscontains an X chromosome, while the sperm carries either an X or Ychromosome.

Distinguishing male-producing from female-producing sperm is most easilyaccomplished by exploiting the difference in the size of the two sexchromosomes. The X chromosome contains more DNA than does the Ychromosome. For example, the difference in total DNA between X-bearingsperm and Y-bearing sperm is 3.4% in boar, 3.8% in bull, and 4.2% in ramsperm.

Distinguishing Cells

To illuminate the workings of cells or distinguish cells that differfrom each other by the slightest difference (e.g., expression of aparticular molecule), various visualization methods have been used fordecades, from simple light microscopic observations to high-voltageelectronic microscopy. In most of these techniques, cells or tissue arepreserved, usually using a cross-linking agent such as an aldehyde(proteins, e.g., glutaraldehyde and formaldehyde), osmium (lipids) or byprecipitating parts of the cells, such as cold methanol and proteins.These techniques suffer from the preparation processes that allow forthe visualization. Fixation procedures often incur artifacts; forexample, in the early days of electronic microscopy (EM), multilamellarbodies were observed but were later understood to be mostly by-productsof the fixation protocols, not actual structures found in livingmammalian cells. While fixation protocols do preserve some of the cellstructure, there are many structures that are difficult to preserve, orwhen preserved under appropriate conditions, the rest of the cellarchitecture is destroyed. Classically, this has been the case for thecytoskeleton, especially for exceptionally dynamic microtubules.

To overcome the limitations of visualization techniques in fixedsamples, “in vivo” approaches have been explored. For example, tounderstand where native polypeptides localize, those polypeptides havebeen purified, associated with a detectable dye (usually covalently),and then introduced into the cell of interest and observed (Chamberlainand Hahn, 2000). This approach does offer the advantages of non-fixedcells; however, the time and expense to purify a target polypeptide,conjugate it to a dye, and then to microinject (a task requiringspecialized equipment, experience, skill and patience) the complex intoa cell often outweigh the advantages. Furthermore, only limited numbersof cells could be examined at any given time due to the limitations ofmicroinjection.

With the advent of the discovery of green and other visible fluorescentproteins (VFPs), however, the ability to visualize polypeptides—evenpolypeptide-polypeptide interactions—became facile and less riddled withartifacts. Green fluorescent protein is a naturally occurringluminescent protein first found in jellyfish. Having been cloned, manyvariants have been produced that produce a rainbow of colors. In mostinstances, the protein of interest is fused by recombinant procedures toa VFP of choice and the trasgene introduced and expressed in the cell ofinterest (Chamberlain and Hahn, 2000). While this approach is farsuperior to previous methods, many extra, time-consuming, steps arerequited from identifying the protein of interest to actuallyvisualizing it in a living cell.

Going beyond cellular localization and movement of proteins, other dyeshave been exploited to identify other processes or stain specificmolecules. For example, calcium-mediated signaling is monitored inliving cells using the fura series of dyes. Other fluorescent dyes havebeen used to test the molecular size barriers of gap junctions in, forexample, epithelial cells. Finally, other stains target specificmolecules, such as double-stranded deoxyribonucleic acid (DNA); suchstains include some of the Hoechst series of dyes, propidium iodide andethidium bromide.

In each case, however, the challenge of introducing the dye or staininto a living cells to the appropriate target area is hindered by thecell membrane which provides a barrier to cells from the outside world.In many cases, dyes are membrane impermeant due to their hydrophobicnature or their size; even membrane-permeant dyes can require longincubation times to breach the membrane and reach the target moleculesor cellular compartments. Breaching the barrier requires a physicalperturbation of the membrane, such as by microinjection or fixation.

Available procedures are few and when available, often faceuncompromising challenges. Even traditional methods of staining DNA incommon methods of sorting sperm cells by gender require extensiveincubation times at elevated temperatures (e.g., 60 minutes at 35° C.;(Johnson, 1992)), permitting quality degradation of the cells. Inaddition, staining must be sufficient so that the signal can beaccurately and precisely detected.

SUMMARY OF THE INVENTION

In a first aspect, the invention discloses methods for staining cells,such as sperm, including bovine sperm, wherein the sperm are mixed witha dye of choice and then electroporating them to facilitate theintroduction of the dye. The sperm can be incubated at temperatures thatenhance sperm viability, typically equal to or less than 39° C.

In a second aspect, the invention discloses methods for sorting cells,such as sperm, by distinguishing differences in DNA content. The cellsare stained with a DNA specific dye by mixing the sperm with the dye andthen electroporating them. The sperm can be maintained at temperaturesthat enhance sperm viability, typically equal to or less than 39° C. Thesperm are then passed before an excitation light source causing thestained DNA to fluoresce, and then passed through means for detectingthe fluorescence and a means for cell sorting, wherein the cells aresorted by DNA content, and the sorted sperm collected. The methods andapparatus are appropriate for mammalian sperm sorting, such as thosefrom bovine, swine, rabbit, alpaca, horse, dog, cat, ferret, rat, mouseand buffalo. Both membrane permeant and impermeant dyes can be used.Useful dyes include those from the SYTOX blue, orange and green series,cyanine dimers and monomers, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1,POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, PO-PRO-1, BO-PRO-1, YO-PRO-1,TO-PRO-1, JO-PRO-1, PO-PRO-3, LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3,TO-PRO-5, acridine homodimer, 7-amino actinomycin D, ethidium bromide,ethidium homodimer-1, ethidium homodimer-2, ethidium nonazide, nuclearyellow, propidium iodide. Other useful dyes include those from SYTO 40blue, green, orange and red fluorescent dyes, Hoechst dyes anddihydroethidium. To enhance the signal, nanoparticles, such as quantumdots and metallic nanoparticles, can be introduced. The particles can betagged with targeting molecules. Sorting efficiency can be greater than90%, while sperm viability rates are greater than 30%, typically greaterthan 90%. Alternatively, instead of electroporating the cells, the dyeis introduced into the cells by osmotic gradients. Cells are firstincubated in hypertonic conditions, and then transferred to hypotonicconditions; the DNA-staining dye can be added to either, or both,hypertonic and hypotonic solutions. After dying the cells, they areready to be sorted or further processed.

In a third aspect, the invention provides methods to pre-select the sexof a mammalian offspring, where the sperm are sorted according to themethods of the invention, and then inseminating a female animal of thesame species as the male animal that provided the sperm. In a fourthaspect, instead of inseminating a female animal, an egg from a femaleanimal is fertilized in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of an electroporation cell

FIG. 1B shows a schematic of a Resistance-Capacitator circuit suitablefor electroporation.

FIG. 2 outlines the steps for collecting, sorting and freezing bovinesperm.

DETAILED DESCRIPTION

While the present invention is susceptible of embodiment in manydifferent forms, there are shown in the drawings and described herein indetail specific embodiments thereof with the understanding that thepresent disclosure is to be considered as an exemplification of theprinciples of the invention and is not intended to limit the inventionto the specific embodiments illustrated.

The present invention solves the problem of breaching the cell membranebarrier to introduce cellular stains and nanoparticles without killingthe cells. Using the methods of the invention, populations of millionsof living cells can be simultaneously stained and are appropriate forfurther applications that require living healthy cells because onceapplied, staining is immediate.

In some embodiments of the invention, electroporation or osmoticgradients are used to permeabilize cell membranes. Electroporationpasses an alternating or direct current (AC or DC) electric field overthe cells. The electric shock blasts holes into the cell membrane. Undercontrolled conditions, the size of the holes are big enough to allowintroduction of the stain and/or nanoparticles, but small enough toprevent excessive cytosol leakage and irreparable cell damage thatresults in cell death. Alternatively, osmotic pressure gradients areused to partially dehydrate the cells. Cells are incubated in ahypertonic solution and then transferred to a hypotonic solution; ineither, or both solutions, the dye is added. As the cells reach osmoticequilibrium with the solution, water flows into the cell, drawing in thedye across the cell membrane.

The methods of the invention also provide the unexpected result ofhastening the diffusion of membrane permeant dyes into cells.

In addition to introducing stains and dyes into living cells, themethods of the invention also allow the introduction of nanoparticlesthat can be used as detectable entities in and of themselves (e.g.,quantum dots) or to amplify a signal, whether innate to a targetmolecule or introduced. For example, metallic nanoparticles createsurface-enhanced resonances, amplifying the natural fluorescence,auto-fluorescence, or fluorescently stained molecules by orders ofmagnitude. Using metallic nanoparticles therefore act as molecularmirrors, deflecting and augmenting available light signals to which theyare in close proximity. The nanoparticles prevent energy loss of thestimulating radiation to other modes, like phonons, and ensure that theenergy is channeled into emitted light. Because the natural fluorescenceintensity of some target molecules, such as DNA, is normally very low,amplification the available signal reduces reliance on dyes or stainswhich can interfere with normal functioning of the target molecule. Forexample, many DNA-specific dyes intercalate between the bases; thisintercalation can, in mitotically or meiotically active cells, introducemutations into the genetic code.

Since the methods of the invention allow for fast live-cell staining,other procedural parameters can be optimized to enhance cell viability.For example, the time during which the cells are mixed with the dye canbe reduced or even eliminated, conserving cellular resources. Thetemperatures at which the cells are manipulated and held can also bereduced, effectuating slower cellular metabolism that again conservescellular resources.

The methods are especially appropriate for sorting sperm by gender, inwhich quick staining of the sperm avoids the problems of reducedviability because of prolonged incubation times.

Definitions

Cell-membrane-rupturing-force means force that is sufficient to disrupta cell membrane such that a cell-impermeant molecule is able to crossthe membrane. In the case of cell membrane-permeant molecules, adisrupted membrane permits faster diffusion of the molecule into thecell.

Comparatively high cell viability rate means a rate wherein at least 5%of the total cell population (e.g., a population of sperm) are alive.The rate can be determined by typical viability tests, includingexclusion of membrane impermeant dyes (e.g., trypan blue), or formonitoring for a specific cellular activity, such as sperm locomotion.

DNA-staining dye means a detectable substance that interacts with apolynucleotide such that when examined under appropriate conditions, thepolynucleotide is optically detected. While most DNA-staining dyesinteract directly with polynucleotides (such as Hoechst stains),DNA-staining dyes also encompass those substances that interact withmolecules that interact with polynucleotides, such as those that bindDNA-binding proteins, such as transcription factors and histones. Insome instances, DNA-staining dye molecules consist of more than onemolecule, such as an antibody tagged with a detectable substance, theantibody specifically binding, for example, a DNA-binding protein.

Electroporation means a phenomenon in which the membrane of a cell,exposed to short, high intensity electric field pulses, is temporarilydestabilized in specific regions of the cell. During the destabilizationperiod, the cell membrane is highly permeable to exogenous moleculespresent in the surrounding media. Electroporation is one method ofproviding a cell membrane-rupturing force.

Hypertonic condition means a condition in which the concentration ofelectrolyte is above that found in cells in the same solution. In thissituation, osmotic pressure leads to the migration of water from thecells to the surrounding solution in an attempt to equalize theelectrolyte concentration inside and outside the cell.

Hypotonic condition means a condition in which the concentration ofelectrolyte is below that found in cells in the same solution. In thissituation, osmotic pressure leads to the migration of water into thecells in an attempt to equalize the electrolyte concentration inside andoutside the cell.

Nonmetallic particle means a nano-scale structure consisting of one ormore metals, such as gold, silver, etc.

Permeating and related terms means to breach a cell membrane. Permeatingthe cell membrane can be accomplished by electroporation and osmoticstress, just as two examples.

Quantum dot means a nano-scale crystalline structure, usually made fromcadmium selenide, and absorbs white light and then re-emits it a coupleof nanoseconds later in a specific color. The size of a quantum dotvaries within the 10⁻⁹ m range, but a quantum dot, regardless of size,is recognizable in that the addition or subtraction of an electronrepresents a significant change in the particle.

Targeting molecule means a molecule that has an affinity for anothermolecule or group of molecules. Examples include antibodies,streptavidin, avidin, biotin, etc.

Making and Using the Invention

Electroporation

When a short, high-voltage pulse surpasses the capacitance of a cellmembrane, transient—and reversible—disruption of a cell membrane occurs(Gehl, 2003). This disruption allows for easier diffusion of smallmolecules into the cell, as well as for electrophoretically drivingmolecules through the destabilized membrane (Gehl, 2003). Anyelectroporation (an electroporator) or other cellmembrane-rupturing-force device which parameters can be manipulated asnecessary by the user can be used in the methods of the invention.Examples include the CUY21EDIT Square Wave Electroporator andSONITRON2000 Sonoporator from Nepa Gene (Ichikawa, Chiba; Japan); theEasyjecT Plus, EasyjecT Optima and EasyjecT-Prima (Flowgen; Nottingham,United Kingdom); Gene Pulser Xcell System™ and MicroPulserElectroporator™ (BioRad Laboratories; Hercules, Calif.). Typically, theresistance values range from 2-10,000 ohms (Ω) depending primarily onthe electrical conductivity of the buffer. The capacitance varies from0.1 millifarads (mF) to 1000 mF.

One embodiment of a suitable electroporation device is shown in FIG. 1.Referring to FIG. 1A, the electroporation device consists of twoparallel glass slides 101 coated with 1,500-2000 angstroms (Å) of indiumtin oxide, which are separated by fragments of number 0 glass coverslips 102, yielding a slide separation of 100 millimeters (mm).Referring now to the circuit in FIG. 1B, the sample cell 103 isconnected to a resistor-capacitator (RC) circuit by alligator clips. Adirect current (DC) power supply 104 is used to charge a capacitor 105.When a switch is thrown, the discharging capacitor generates atime-dependant and spatially uniform electric field across the sample.An oscilloscope is used to monitor the voltage across the sample cell asa function of time. The RC circuit formed by the sample cell andcapacitor allow for a well-controlled electric field to be generated.The resistance (R) of the circuit is left floating—that is, determinedby the geometry and content of the sample cell.

The temperature at which the cells are subjected to electroporationvaries with the cell type and the intended application. For example, inthe case of staining mammalian sperm cells for sorting by gender, suchas those from bovines, a temperature of about −4° C. to about 39° C.;preferably about 0° C. to about 25° C., more preferably about 0° C. toabout 12° C., and most preferably about 0° C., 1° C., 2° C., 3° C., 4°C., 5° C. and 6° C.

Cells are suspended in an osmotically appropriate buffer forelectroporation, although the solution can be hyper- or hypotonic toincrease the efficiency of electroporation. For example, a 0.35 Msucrose solution yields good results for bovine sperm. Appropriatebiological buffers include Hank's Balanced Salt Solution (HBSS), sodiumphosphate-based buffers, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonicacid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane(BIS-Tris), N-(2-hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPSor HEPPS), glyciclycine, N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonicacid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS),piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate,3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid)TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-glycine (Tricine), andtris(hydroxymethyl)-aminomethane (Tris). Salt solutions that can be usedto produce osmotically appropriate conditions include Alsoverr'sSolution, Dulbecco's Phosphate Buffered Saline (DPBS), Earle's BalancedSalt Solution, Gey's Balanced Salt Solution (GBSS), Puck's Saline A,Tyrode's Salt Solution, St. Thomas Solution and University of WisconsinSolution. In some instances, a simple sucrose solution is sufficient; inothers, a simple buffer.

Because cells differ from organism to organism, of even the same celltype, the parameters for electroporation may need to be determinedexperimentally. An assay is provided to determine the appropriateparameters for each cell type.

Assay to Determine Parameters for Electroporation Introduction of Dyesand Nanoparticles

Cells are harvested according to established procedures, preferably at4° C. or other metabolic-suspending temperatures, washed, andre-suspended in an osmotically appropriate buffer, preferably withoutadditional divalent cations, such as Ca²⁺, Mg²⁺ and Mn²⁺, at aconcentration of 1-2×10⁷ cells per ml. The concentration of the cellscan be altered to accommodate the differences in cell size and othervariables. The desired dye and/or nanoparticles are added to thesuspension; the concentrations of which can be experimentallydetermined. In the case of dyes wherein the binding sites are known(e.g., intercalation between adenosine and threonine in DNA), anestimate of the appropriate dye concentration can be calculated by usingan estimate of the amount of DNA in the sample. In some cases, thecells, dye and/or nanoparticles are incubated in the electroporationcell for a short period of time, e.g., 1-15 minutes, preferably 1-5minutes, preferably less than 5 minutes, more preferably less than 1minute, and most preferably 30 seconds at a metabolically-suspendingtemperature (e.g., 0-4° C.). In other instances, there is nopre-incubation and the cells are electroporated immediately. In the caseof nanoparticles, incubation times are minimized to prevent any settlingor non random dispersion of the particles. Alternatively, the viscosityof the buffer can be altered to maintain the suspension of particles,such as the addition of a protein (e.g., bovine serum albumin) or inertsubstance.

An electric pulse is applied; a starting voltage of 2.0 kV represents areasonable starting point, with the current set at a maximum of 0.9 mA.Adjustable current and wattage dials are set at bare minimum. In somecases, the cells, dye and/or nanoparticles are incubated in theelectroporation cell for a short period of time after electroporation,e.g., 1-15 minutes, preferably 1-5 minutes, preferably less than 5minutes, more preferably less than 1 minute, and most preferably 30seconds at a metabolically-suspending temperature (e.g., 0-4° C.). Inother instances, no post-incubation step is necessary. For other celltypes, restoring physiological conditions is paramount to preserve cellviability; for these cells, a recovery solution (e.g., culture media) isadded immediately. Cells are then transferred for further processing(e.g., washing, collecting, freezing) and observation. In most cases, arecovery media containing divalent cations is preferable, such asprovided in appropriate growth media.

Table 1 presents just one example of an experimental design (adaptedfrom (Potter et al., 1984)); this example is not meant to be limiting.One of skill in the art will know how to manipulate these and otherappropriate experimental parameters.

TABLE 1 Example of experimental parameters for determining transferfrequency by electroporation Power supply Cells settings (kV/mA)Electrode Temperature (° C.) A 1.2/300 Al 20 A 1.2/300 Al 20 Temperatureand voltage effects A 1.2/300 Al 20 A 1.2/300 Al 0 A 1.2/300 Al 20 A4.0/0.9 Al 20 A 4.0/capacitor Al 20 A 1.2/100 SS 20 A 1.2/100 ss 0 A1.2/300 SS 0 A 2.0/0.9 ss 0 A 4.0/capacitor ss 0 Comparison of celllines and species A 4.0/0.9 Al 0 B 4.0/0.9 Al 0 C 4.0/0.9 Al 0 D 4.0/0.9Al 0

For bovine sperm, when introducing macromolecules such as DNA, a pulseof 0.25 seconds at 25 μF capacitance and 300 V is sufficient in a 1.4 mlelectroporation chamber where the electrodes are 4 mm apart (Rieth etal., 2000). For smaller molecules, shorter pulses (e.g., approximately0.25 ms) at lower voltages (e.g., approximately 10 V) can be used (seeExample 1).

Cells

Cells or tissue samples that are appropriate for the methods of theinvention are collected from a subject or a culture. The subject can bea vertebrate, more preferably a mammal, such as a bull, monkey, dog,cat, rabbit, pig, goat, sheep, horse, rat, mouse, guinea pig, etc. Anytechnique to collect the desired cells may be employed, includingbiopsy, surgery, scrape (inner cheek, skin, etc.), induced ejaculation(for sperm) and blood withdrawal. Any cultured cell type, whether exvivo cultured cells from a subject, or a cell line, such as Madin-DarbyCanine Kidney (MDCK), HeLa, CaCO-2, immunoglobulin-secreting hybridomas,etc. can also be used in the methods of the invention.

Stains, Dyes and Other Visual Labels

To detect a molecule of interest, a label can be used. The label can becoupled to a binding antibody or other interacting polypeptide, or toone or more particles, such as a nanoparticle. Suitable labels includefluorescent moieties, such as fluorescein isothiocyanate; fluoresceindichlorotriazine and fluorinated analogs of fluorescein;naphthofluorescein carboxylic acid and its succinimidyl ester;carboxyrhodamine 6G; pyridyloxazole derivatives; Cy2, 3 and 5;phycoerythrin; fluorescent species of succinimidyl esters, carboxylicacids, isothiocyanates, sulfonyl chlorides, and dansyl chlorides,including propionic acid succinimidyl esters, and pentanoic acidsuccinimidyl esters; succinimidyl esters of carboxytetramethylhodamine;rhodamine Red-X succinimidyl ester; Texas Red sulfonyl chloride; TexasRed-X succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester;Red-X; Texas Red dyes; tetramethylrhodamine; lissamine rhodamine B;tetramethylrhodamine; tetramethyrhodasmine isothiocyanate;naphthofluoresceins; coumarin derivatives; pyrenes; pyridyloxazolederivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuranisothiocyanates; sodium tetrafluorophenols;4,4-difluoro-4-bora-3a,4a-dia-za-s-indacene. In some cases enzymaticmoieties can be appropriate, such as alkaline phosphatase or horseradishperoxidase; and radioactive moieties, including ³⁵[S] and ¹³⁵[I] labels.The choice of the label depends on the application, the desiredresolution and the desired observation methods. For fluorescent labels,the fluorophore is excited with the appropriate wavelength, and thesample observed using a microscope, confocal microscope, orfluorescence-activate cell sorting (FACS) machine. In the case ofradioactive labeling, the samples are contacted with autoradiographyfilm and developed; alternatively, autoradiography can also beaccomplished using ultrastructural approaches.

Dyes and stains that are specific for DNA (or preferentially bind doublestranded polynucleotides in contrast to single-stranded polynucleotides)include Hoechst 33342(2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole)and Hoechst 33258(2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole)and others of the Hocchst series; SYTO 40, SYTO 11, 12, 13, 14, 15, 16,20, 21, 22, 23, 24, 25 (green); SYTO 17, 59 (red), DAPI, YOYO-1,propidium iodide, YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3, SYTOX Green,SYTOX, methyl green, acridine homodimer, 7-aminoactinomycin D,9-amino-6-chloro-2-methoxyactridine. Tables 1, 2 and 3 list many of theavailable polynucleotides-specific/chromosome specific stains currentlyavailable (Tables 2-4 have been adapted from (Haugland, 2002)).

TABLE 2 Cell membrane-impermeant cyanine nucleic acid stains Catalogue#¹ Dye Name Ex/Em* Excitation Source† SYTOX Dyes S11348 SYTOX Blue445/470 Hg-arc lamp, 436 nm line S7020 SYTOX Green 504/523 Ar-ion laser,488 nm line S11368 SYTOX Orange 547/570 Nd:YAG laser, 532 nm lineCyanine Dimers P3580 POPO-1 434/456 Hg-arc lamp, 436 nm line He—Cdlaser, 442 nm line B3582 BOBO-1 462/481 Hg-arc lamp, 436 nm line He—Cdlaser, 442 nm line Y3601 YOYO-1 491/509 Ar-ion laser, 488 nm line T3600TOTO-1 514/533 Ar-ion laser, 514 nm line J11372 JOJO-1 529/545 Nd:YAGlaser, 532 nm line P3584 POPO-3 534/570 Nd:YAG laser, 532 nm line L11376LOLO-1 565/579 Kr-ion laser, 568 nm line B3586 BOBO-3 570/602 Hg-arclamp, 578 nm line Y3606 YOYO-3 612/631 Orange He—Ne laser, 594 nm lineT3604 TOTO-3 642/660 He—Ne laser, 633 nm line Kr-ion laser, 647 nm line635 nm diode laser Cyanine Monomers P3581 PO-PRO-1 435/455 Hg-arc lamp,436 nm line He—Cd laser, 442 nm line B3583 BO-PRO-1 462/481 Hg-arc lamp,436 nm line He—Cd laser, 442 nm line Y3603 YO-PRO-1 491/509 Ar-ionlaser, 488 nm line T3602 TO-PRO-1 515/531 Ar-ion laser, 514 nm lineJ11373 JO-PRO-1 530/546 Nd:YAG laser, 532 nm line P3585 PO-PRO-3 539/567Nd:YAG laser, 532 nm line He—Ne laser, 543 nm line L11377 LO-PRO-1567/580 Kr-ion laser, 568 nm line B3587 BO-PRO-3 575/599 Hg-arc lamp,578 nm line Y3607 YO-PRO-3 612/631 He—Ne laser, 594 nm line T3605TO-PRO-3 642/661 He—Ne laser, 633 nm line Kr-ion laser, 647 nm lineT7596 TO-PRO-5 747/770 Laser diodes ¹According to (Haugland, 2002),catalogue numbers are specific to Molecular Probes, Inc. *Wavelengths ofexcitation (Ex) and emission (Em) maxima, in nm. †Nearest major emissionline of some common light sources.

TABLE 3 Cell-permeant cyanine nucleic acid stains Catalogue #¹ Dye Name*Ex/Em† Blue-fluorescent SYTO dyes S11351 SYTO 40 blue-fluorescentnucleic acid stain 419/445 S11352 SYTO 41 blue-fluorescent nucleic acidstain 426/455 S11353 SYTO 42 blue-fluorescent nucleic acid stain 430/460S11354 SYTO 43 blue-fluorescent nucleic acid stain 437/464 S11355 SYTO44 blue-fluorescent nucleic acid stain 445/472 S11356 SYTO 45blue-fluorescent nucleic acid stain 452/484 Green-fluorescent SYTO DyesS34854 SYTO 9 green-fluorescent nucleic acid stain 483/503 S32704 SYTO10 green-fluorescent nucleic acid stain 484/505 S34855 SYTO BCgreen-fluorescent nucleic acid stain 485/500 S7575 SYTO 13green-fluorescent nucleic acid stain 488/509 S7578 SYTO 16green-fluorescent nucleic acid stain 488/518 S7559 SYTO 24green-fluorescent nucleic acid stain 490/515 S7556 SYTO 21green-fluorescent nucleic acid stain 494/517 S32706 SYTO 27green-fluorescent nucleic acid stain 495/537 S32705 SYTO 26green-fluorescent nucleic acid stain 497/534 S7558 SYTO 23green-fluorescent nucleic acid stain 499/520 S7574 SYTO 12green-fluorescent nucleic acid stain 500/522 S7573 SYTO 11green-fluorescent nucleic acid stain 508/527 S7555 SYTO 20green-fluorescent nucleic acid stain 512/530 S7557 SYTO 22green-fluorescent nucleic acid stain 515/535 S7577 SYTO 15green-fluorescent nucleic acid stain 516/546 S7576 SYTO 14green-fluorescent nucleic acid stain 517/549 S7560 SYTO 25green-fluorescent nucleic acid stain 521/556 Orange-fluorescent SYTOdyes S32707 SYTO 86 orange-fluorescent nucleic acid stain 528/556 S11362SYTO 81 orange-fluorescent nucleic acid stain 530/544 S11361 SYTO 80orange-fluorescent nucleic acid stain 531/545 S11363 SYTO 82orange-fluorescent nucleic acid stain 541/560 S11364 SYTO 83orange-fluorescent nucleic acid stain 543/559 S11365 SYTO 84orange-fluorescent nucleic acid stain 567/582 S11366 SYTO 85orange-fluorescent nucleic acid stain 567/583 Red-fluorescent SYTO dyesS11346 SYTO 64 red-fluorescent nucleic acid stain 598/620 S11343 SYTO 61red-fluorescent nucleic acid stain 620/647 S7579 SYTO 17 red-fluorescentnucleic acid stain 621/634 S11341 SYTO 59 red-fluorescent nucleic acidstain 622/645 S11344 SYTO 62 red-fluorescent nucleic acid stain 649/680S11342 SYTO 60 red-fluorescent nucleic acid stain 652/678 S11345 SYTO 63red-fluorescent nucleic acid stain 654/675 ¹According to (Haugland,2002), catalogue numbers are specific to Molecular Probes, Inc.†Wavelengths of excitation (Ex) and emission (Em) maxima, in nm.

TABLE 4 Properties of classic nucleic acid stains Fluorescence Catalogue#¹ Dye Name Ex/Em* Emission Color Applications† A666 Acridine homodimer431/498 Green Impermeant AT-selective High-affinity DNA binding A13107-AAD (7-amino- 546/647 Red Weakly permeant actinomycin D) GC-selectiveFlow cytometry Chromosome banding A1324 ACMA 419/483 Blue AT-selectiveAlternative to quinacrine for chromosome Q banding D1306, D3571, DAPI358/461 Blue Semi-permeant D21490 AT-selective Cell-cycle studiesChromosome and nuclei counterstain Chromosome banding D1168, D11347,Dihydroethidium 518/605 Red§ Permeant D23107 Blue fluorescent untiloxidized to ethidium E1305, E3565‡ Ethidium bromide 518/605 RedImpermeant dsDNA intercalator Dead-cell stain Chromosome counterstainFlow cytometry Argon-ion laser excitable E1169 Ethidium homodimer-1528/617 Red Impermeant (EthD-1) High-affinity DNA labeling Dead-cellstain Argon-ion and green He—Ne laser excitable E3599 Ethidiumhomodimer-2 535/624 Red Impermeant (EthD-2) Very high-affinity DNAlabeling Electrophoresis prestain E1374 Ethidium monoazide 464/625 RedImpermeant (unbound)** Photocrosslinkable H1398, H3569‡, Hoechst 33258(bis- 352/461 Blue Permeant H21491 benzimide) AT-selective Minorgroove-binding dsDNA-selective binding Chromosome and nuclearcounterstain H1399, H3570‡, Hoechst 33342 350/461 Blue Permeant H21492AT-selective Minor groove-binding dsDNA-selective binding Chromosome andnuclear counterstain H21486 Hoechst 34580 392/498 Blue PermeantAT-selective Minor groove-binding dsDNA-selective binding Chromosome andnuclear counterstain H22845 Hydroxystilbamidine 385/emission variesVaries AT-selective with nucleic acid Spectra dependent on secondarystructure and sequence RNA/DNA discrimination L7595 LDS 751 543/712(DNA) Red/infrared Permeant 590/607 (RNA) High Stokes shiftLong-wavelength spectra Flow cytometry N21485 Nuclear yellow 355/495Yellow Impermeant Nuclear counterstain P1304MP, Propidium iodide (PI)530/625 Red Impermeant P3566‡, P21493 Dead-cell stain Chromosome andnuclear counterstain ¹According to (Haugland, 2002), catalogue numbersare specific to Molecular Probes, Inc. *Excitation (Ex) and emission(Em) maxima in nm. †Indication of dyes as “permeant” or “impermeant” arefor the most common applications; permeability to cell membranes mayvary considerably with the cell type, dye concentrations and otherstaining conditions. §After oxidation to ethidium. **Prior tophotolysis; after photolysis the spectra of the dye/DNA complexes aresimilar to those of ethidium bromide-DNA complexes.

In some cases, such is DNA and certain polypeptides, a physicalcharacteristic of that molecule can be used, such as the innateautofluorescence in DNA. In such cases, signal intensity can bemodulated by the introduction of nanoparticles (see Modulatingfluorescence signals with nanoparticles, below).

Introducing Dye Via Osmolarity/Osmolality Modulation

The modulation of the concentration of solutes can create an environmentthat is either hypertonic or hypotonic to cells. By suspending the cellsin a hypertonic solution, cells become partially dehydrated. After ashort period, they are then transferred to a hypotonic solution. Either,or both solutions can include the dye of interest, but should be presentto be available to the cells to enter the cells. Preferably, the dye ispresent in at least the hypotonic solution. As the cells reach osmoticequilibrium with the solution, water flows into the cell, drawing in thedye across the cell membrane.

Osmolality can be varied by either adding appropriate salts or othersolutes that are compatible with the cells of interest (e.g., KCl, NaCl,MgCl₂, MnCl₂, CaCl₂, sucrose, glucose, etc.), or by diluting thesolution with water or buffer. After collection, cells are transferredto a hypertonic solution for about 0 to about 15 minutes, preferably,about 1 to about 10 minutes, more preferably about 3 to about 7 minutes,and most preferably about 5 minutes. The temperature of the solution isabout −4° C. to about 39° C., preferably about 0° C. to about 25° C.,more preferably 0° C. to about 12° C., and most preferably, about 4° C.The cells are then transferred to a hypotonic solution or the solutionin which the cells are in is diluted with buffer to create hypotonicconditions. The temperature of the added solution is bout −4° C. toabout 39° C., preferably about 0° C. to about 25° C., more preferably 0°C. to about 12° C., and most preferably, about 4° C.

Osmolality conditions vary somewhat by cell type. However, for bovinesperm, hypertonic conditions are created at approximately greater than250 mOsm, whereas hypotonic conditions are created at approximately lessthan 250 mOsm (Liu and Foote, 1998). Preferred hypertonic osmalitiesinclude 100 mOsm to 249 mOsm; most preferably greater than 150 mOsm, butless than 250 mOsm. Hypotonic osmalities include 251 mOsm to 1537 mOsm;preferably 500 mOsm to 963 mOsm; and most preferably greater than 250mOsm but less than 732 mOsm. The dye can be any of those listed inTables 2-4 or other appropriate dye.

Modulating Fluorescence Signals with Nanoparticles (Quantum Dots andMetallic Nanoparticles)

Organic and biomolecular fluorophores generally exhibit only moderateStokes shifts between their excitation and emission spectra, haverelatively broad emission spectra, and photobleach when monitored overextended periods of time. A promising alternative to conventionalfluorophores is quantum dots (QDs) (Doty et al., 2004).

In one embodiment, the core of a QD consists of a semiconductornanocrystal, such as CdSe, surrounded by a passivation shell, such asZnS. Upon absorption of a photon, an electron-hole pair is generated,the recombination of which in ˜10-20 ns leads to the emission of aless-energetic photon. This energy, and therefore the wavelength, isdependent on the size of the core (smaller->lower wavelength), which canbe varied almost at will by controlled-synthesis conditions (Lidke andAmdt-Jovin, 2004). The surface is coated with a polymer that protectsthe QD from water and allows for chemical coupling to molecules.

The excitation spectra of QDs are a continuum, rising into theultraviolet, and the emission spectra are narrow and slightlyred-shifted to the band-gap absorption. Thus QDs with differentemissions can be excited with a single excitation (Smith and Nie, 2004).The large extinction coefficient and the relatively high quantum yieldof QDs, as well as their extraordinary photostability, permit the use ofa low sample irradiance and prolonged imaging with a detectionsensitivity extending down to the single-QD level.

QDs are commercially available (e.g., Quantum Dot Corp.; Hayward, Calif.and Evident Technologies; Troy, N.Y.) with a variety of conjugated orreactive surfaces, e.g., amino, carboxyl, streptavidin, protein A,biotin, and immunoglobulins. QDs are non-toxic to most cells. Forexample, tissue culture cells loaded with QDs survive for weeks withoutdiminished growth or division, and the QDs persisted the entire time(Doty et al., 2004). In live animal studies, mice lived normal liveswith QDs for months without obvious deleterious effects (Lidke andAmrdt-Jovin, 2004). QDs can be introduced into sexual reproductive cellswithout harm. For example, Xenopus embryos injected with QDs did notalter the subsequent phenotype; the QDs were viewable throughoutdevelopment (Smith and Nie, 2004).

QDs can be targeted to specific areas of the cell, such as the nucleus,by coating them with appropriate molecules, such as DNA-bindingmolecules (oligonucleotides, DNA-binding proteins, such as histones,transcription factors, polymerases and other molecules of the chromatin,DNA-binding dyes, such as those listed in Tables 2-4, or other smallmolecules, such as other base intercalators). The particles aresuspended with the cells prior to electroporation.

Similarly, metallic nano-particles can be used to enhance anyfluorescent signal, such as those made of gold and silver. They canlikewise be tagged with targeting molecules such that they are in closeproximity of the stained DNA.

Detecting Chromosomal Differences with Nanotransistors andPhoto-Activatable Fluorohores

The ability to incorporate an indicator that has a strongly non-linearresponse to DNA amount facilitates measuring DNA content. Ideally, thisindicator has very little or no fluorescence for the amount of DNAassociated with one chromosome, and large amounts of fluorescence forthe amount of DNA associated with another chromosome, although inpractice approximations to this non-linear response curve are extremelyuseful. There are several mechanisms that exist for generating suchnon-linear fluorescence. Photo-activated fluorophores are one suchmechanism. Incorporating a photo-activated fluorophore which has anon-linear response to the usual fluorescence emitted by a DNA stainprovides a flexible combination of fluorophores whose properties may betuned to achieve the desired non-linearity (Hogan, 2005). Suchphoto-activated fluorophores would not necessarily have to beincorporated into the nucleus of the sperm as they may be tuned torespond to fluorescence from the sperm as a whole. By incorporatingmolecular or nano-transistors into the medium or the sperm, they act asnon-linear amplifiers for fluorescence radiated from DNA, either innateautofluorescence or that generated from a stain. Such nano-transistorshave countless embodiments because they can be based on biologicalproteins, or based on quantum dot transistors. There is a significantadvantage in that the light that is measured is provided by a strong“pumping” source as opposed to the weak gating source that is usuallyassociated with natural fluorescence of DNA or staining, like Hoeschtstains and others listed in Tables 2-4.

Metallic Nanoparticles and Other Modifiers of Fluorophore Free-SpaceSpectral Properties

Nearby conducing metallic particles, colloids or surfaces can modifyfree-space spectral conditions of fluorophores such that the incidentelectric field “felt” by the fluorophore is increased (or decreased),and the rate of radiavity decay can also be modulated (Asian et al.,2004). The radiavity decay rate is that at which a fluorophore emitsphotons. Because the metallic nanoparticles need to be in closeproximity to the fluorescent molecule (approximately about 5 nm),particles can be tagged with fluorescent molecules; or, in the case ofpolynucleotides (which have a low level of auto fluorescence at 260 nmand 280 nm), tagged with molecules that bind the polynucleotides, suchas oligonucleotides, small molecules, or polynucleotide specific bindingpolypeptides. The particles are suspended with the cells prior toelectroporation.

Flow Cytometry/Fluorescence-Activated Cell Sorting (FACS)

Methods of performing flow cytometry are well known (Lidke andArndt-Jovin, 2004). Flow cytometry (measurement of cells as they flow bya detector) has been available for analysis and sorting a variety ofcell types in fluid suspension since the late 1970s. Flow cytometers usefocused laser light to illuminate cells as they pass the laser beam, oneat a time, in a fine fluid stream. Light scattered by the cells andlight emitted by fluorescent dyes attached or loaded in the cells areanalyzed by detectors. Cells can be distinguished and selected on thebasis of size and shape, as well as by the presence of differentmolecules inside and on the surface of the cells.

FIG. 2 outlines the flow that can be applied to gender-sorting of sperm.The sperm are collected from the donor 201 and subjected to extension202 and then cooled slowly to 6° C. Once cooled, the sperm can besubjected to staining DNA by any technique, but preferably the noveltechniques of the present invention, using electroporation or osmolalityand/or nanoparticles of various compositions. The stained cells areintroduced into a cell sorting device 204 and separated based on genderdifference, usually by the sex chromosomes X and Y. The sorted cells arecollected, and slowly cooled to 4° C. 205 before being subjected to afinal extension 206. The cells are loaded into cryogenic-compatiblestraws 207, the cell allowed to settle 208, and then frozen 209.

In the methods of the invention, because of the advantages of stainingcells at 4° C. or even cooler, the cells may be cooled to greater than6° C. as shown in step 203. Sorting itself 204 can also take place atcooler temperatures, thus preserving cell integrity and cell viability.In many bovine sperm separation protocols, eggs, egg yolks or othersperm-supporting substances are added to the collected sperm to improveviability, however, because the present invention allows for stainingand sorting of the cells at cool temperatures-those in which the cellsare metabolically inactive, or nearly so (“metabolically suspended”—sucha step (and the potential of introducing a confounding variable insorting, being obliged to pre-filter before sorting, and potentiallycontaminating the sample with microbes) can be eliminated, or thequantity of added egg or other sperm-supporting substances can bereduced to facilitate sorting and other processing.

Eliminating Dead Cells

After electroporation or osmotic introduction of dyes and/ornanoparticles, a portion of the cell population will usually benonviable. To increase the quality of the output of the cell populationsat the end of processing the cells, dead cells can be removed from thelive cells.

In the case of using fluorescent dyes, such as those that bind DNAlisted in Tables 2-4, dead cells and successfully stained, viable cells,both fluoresce. This is because dead cells have compromised cellmembranes. To eliminate dead cells from the population, one approach isto add a counter-stain that diminishes the signal. For example, insperm, membrane-impermeant red food coloring is mixed with the cells. Byfirst dyeing the sperm with an impermeable dye, the Hoechst dye is notable to bind to the DNA since the impermeable dye is already bound.Therefore, the fluorescence of the dead sperm should be eithereliminated or a different color, depending on the impermeable DNA dyeused. Other classic tests include Trypan blue exclusion, where onlynon-viable cells allow entry of the dye, diminishing fluorescentsignals. In this case, the dye is added after electroporation or osmoticshock, but before sorting.

EXAMPLES

The following example is for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

Example 1: In Vivo Staining of Sperm Cells with a DNA-Specific Dye

Methods and Materials

Electroporation Unit

A sample cell was formed by two parallel glass slides coated with1,500-2000 angstroms (Å) of indium tin oxide (ITO). The slides wereseparated by fragments of number zero glass cover slips, yielding aslide separation of 100 millimeters (mm).

The sample cell was connected to a resistor-capacitator (RC) circuit byalligator clips. A direct current (DC) power supply was used to charge acapacitor. When a switch was thrown, the discharging capacitor generateda time-dependant and spatially uniform electric field across the sample.An oscilloscope was used to monitor the voltage across the sample cellas a function of time.

The RC circuit formed by the sample cell and capacitor allowed for awell-controlled electric field to be generated. The resistance (R) ofthe circuit was left floating—that is, determined by the geometry andcontent of the sample cell. Typical R values ranged from 2-10,000 watts(W) depending primarily on the electrical conductivity of the buffer.The capacitance was varied from 0.1 millifarads (mF) to 1000 mF.

Cells

Bovine sperm that had been previously frozen were thawed for 60 secondsin a 96° F. water bath. Sperm were then centrifuged at 2,000 rotationsper minute (rpm) for two minutes and the supernatant decanted. Spermwere then re-suspended with 0.35 M sucrose to partially dehydrate thesperm. The sperm solution was then incubated at 96° F. for 15 minutesand then transferred to the sample cell of the electroporation unit.

Electroporation

A voltage was applied to the capacitor circuit, and then the powersupply was disconnected. A switch was then thrown and the capacitordischarged across the sample cell. These steps were carried out within15 seconds of transferring the sperm to the sample cell to retain arandom orientation of sperm with respect to the electric field.Electroporation was carried out on sperm in isotonic (0.25 M sucrose)and hypertonic (0.35 M sucrose) solutions. In each case, 10 V wasapplied to the capacitor and a time constant of 0.26 milliseconds (ms)measured.

A solution of 0.1 M sucrose and the DNA-specific dye, propidium iodide,was then injected into the sample cell. Fluorescence microscopy is usedto image the sperm.

Results

Since propidium iodide dye does not breach the cell membrane barrier,only cells that have had their membranes compromised, such as byelectroporation, allow entry of the dye, which then binds to any DNA inthe cell (the nucleus and mitochondria), and, when excited with theappropriate wavelength of light, fluoresces. To distinguish fluorescentdead cells from fluorescent live cells, sperm motility was assessed.

In each case (isotonic and hypertonic/hypotonic solution), motile andfluorescent sperm were observed. However, the number of these sperm wasenhanced by approximately five-ten fold in the hypertonic/hypotonicsolution. Control samples unexposed to electric fields did not yieldmotile and fluorescent sperm.

Motility was examined in the hypertonic/hypotonic solution and comparedto a control sample unexposed to an electric field. The electroporatedsample yielded a loss of 70% of motile sperm compared to the control. Inaddition, the electroporated sample showed a 68% increase in the numberof damaged and non-motile sperm. All of the observed motile sperm in thehypertonic/hypotonic solution exposed to the electric field displayedfluorescence.

Larger field strengths, multiple pulses, alternating current (AC)fields, buffers with larger osmotic pressures, and longer time constantsled to complete loss of sperm motility. Weaker field strengths andshorter time constants did not yield fluorescent motile sperm.

A temporary spatially uniform electric field allows non-permanentmembrane dyes to cross bovine sperm cell membranes. A 70% loss inmotility was associated with this process—but manipulating procedureparameters can reduce the death toll. This technique also allows for theintroduction of nanoparticles into sperm and other membrane bound-cells.

REFERENCES

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1-20. (canceled)
 21. A method for labeling DNA in a plurality of cellsusing a tagged oligonucleotide, comprising: (a) incubating the pluralityof cells in a first solution, at a temperature from about −4° C. toabout 39° C., wherein the first solution is a hypertonic solution havingan osmolality of at least about 250 mOsm; (b) forming a mixturecomprising the plurality of cells, the tagged oligonucleotide, and asecond solution, wherein the second solution is a hypotonic solutionhaving an osmolality of less than about 250 mOsm, and wherein at leastthe second solution comprises a buffer compatible with electroporation;(c) incubating the mixture, at a temperature from about −4° C. to about39° C.; and (d) electroporating the mixture to provide substantiallyuniform labeling of DNA.
 22. The method of claim 21, wherein the taggedoligonucleotide specifically hybridizes to the DNA at a DNA sequencecomplementary to the tagged oligonucleotide, wherein the DNA sequencecomplementary to the tagged oligonucleotide is a selected chromosomalregion, and hybridization of the tagged oligonucleotide and the DNAsequence complementary to the tagged oligonucleotide allows subsequentdetection of the selected chromosomal region.
 23. The method of claim22, wherein the plurality of cells are mammalian.
 24. The method ofclaim 23, wherein the plurality of cells are bovine sperm.
 25. Themethod of claim 24, wherein duration of incubation in the first solutionis about less than 15 minutes.
 26. The method of claim 24, wherein theosmolality of the first solution is between about 500 mOsm and about 730mOsm, and the osmolality of the second solution is between about 150mOsm and 250 mOsm.
 27. The method of claim 24, wherein the taggedoligonucleotide comprises a fluorescent label.
 28. The method of claim27, wherein the tagged oligonucleotide further comprises a quantum dotor a nanoparticle.
 29. The method of claim 27, wherein the fluorescentlabel is selected from the group consisting of fluoresceinisothiocyanate, fluorescein dichlorotriazine, fluorinated analogs offluorescein, naphthofluorescein carboxylic acid, naphthofluoresceincarboxylic acid succinimidyl ester, carboxy rhodamine 6G, pyridyloxazolederivative Cy2, pyridyloxazole derivative 3, pyridyloxazole derivativeC5, phycoerythrin, fluorescent species of Succinimidyl esters,carboxylic acids, isothiocyanates, sulfonyl chlorides, or dansylchlorides, propionic acid Succinimidyl esters, pentanoic acidSuccinimidyl esters, Succinimidyl esters of carboxytetramethylihodamine,rhodamine Red-X succinimidyl ester, Texas Red sulfonyl chloride, TexasRed-X succinimidyl ester, Texas Red-X sodium tetrafluorophenol ester,Red-X: Texas Red dyes, tetramethylrhod amine, lissarnine rhodamine B,tetramethylrhodamine, tetramethylrhodamine isothiocyanate,naphthofluorescein, coumarin derivatives, pyreries, pyridyloxazolederivatives, dapoxyl dyes, Cascade Blue dye, Cascade Yellow dye,benzofuran isothiocyanates, sodium tetrafluorophenols, and4.4-difluoro-4-bora-3a,4a-dia-Za-S-indacene.
 30. The method of claim 24,wherein incubating the mixture prior to electroporation is at a firstmetabolically-suspending temperature for a duration of about less than15 minutes.
 31. The method of claim 30, wherein the firstmetabolically-suspending temperature is about 0° C. to about 4° C. 32.The method of claim 24, further comprising: (e) incubating the mixtureafter electroporation at a second metabolically-suspending temperaturefor a duration of about less than 15 minutes.
 33. The method of claim32, wherein the second metabolically-suspending temperature is about 0°C. to about 4° C.
 34. The method of claim 24, wherein electroporation isa pulse of about 0.25 seconds.
 35. The method of claim 24, wherein thefirst solution comprises a buffer compatible with electroporation. 36.The method of claim 24, wherein the tagged oligonucleotide is present inthe first solution.
 37. The method of claim 21, wherein the taggedoligonucleotide is present in at least the second solution to permeatethe membrane of each of the plurality of cells when osmotic equilibriumwith the second solution is reached.